A High‐Throughput Visual Screen for the Directed Evolution of C β ‐stereoselectivity of L‐threonine Aldolase

L‐Threonine aldolase (L‐TA) is a pyridoxal phosphate‐dependent enzyme that catalyzes the reversible condensation of glycine and aldehydes to form β‐hydroxy‐α‐amino acids. The combination of directed evolution and efficient high‐throughput screening methods is an effective strategy for enhancing the enzyme’s catalytic performance. However, few feasible high‐throughput methods exist for engineering the Cβ‐stereoselectivity of L‐TAs. Here, we present a novel method of screening for variants with improved Cβ‐stereoselectivity; this method couples an L‐threo‐phenylserine dehydrogenase, which catalyzes the specific oxidation of L‐threo‐4‐methylsulfonylphenylserine (L‐threo‐MTPS), with the concurrent synthesis of NADPH, which is easily detectable via 340‐nm UV absorption. This enables the visual detection of L‐threo‐MTPS produced by L‐TA through the measurement of generated NADPH. Using this method, we discover an L‐TA variant with significantly higher diastereoselectivity, increasing from 0.98% de (for the wild‐type) to 71.9% de.